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Like all DNA viruses, human cytomegalovirus (HCMV) infection is known to result in profound effects on host cell cycle. Infection of fibroblasts with HCMV is known to induce an advance in cell cycle through the G(0)-G(1) phase and then a subsequent arrest of cell cycle in early S-phase, presumably resulting in a cellular environment optimum for high levels of viral DNA replication whilst precluding replication of cellular DNA. Although the exact mechanisms used to arrest cell cycle by HCMV are unclear, they likely involve a number of viral gene products and evidence points to the ability of the virus to prevent licensing of cellular DNA synthesis. One viral protein known to profoundly alter cell cycle is the viral immediate early 86 (IE86) protein--an established function of which is to initially drive cells into early S phase but then inhibit cellular DNA synthesis. Here we show that, although IE86 interacts with the cellular licensing factor Cdt1, it does not inhibit licensing of cellular origins. Instead, IE86-mediated inhibition of cellular DNA synthesis requires mini-chromosome-maintenance 3 (MCM3) associated protein (MCM3AP), which can cause subsequent inhibition of initiation of cellular DNA synthesis in a licensing-independent manner.

SummaryCompared to sperm nuclei, nuclei from adult somatic cells replicate inefficiently in frog egg extract. In this issue of Cell, Lemaitre et al. (2005) show that pre-exposure of erythrocyte nuclei to a mitotic extract removes this difference, reorganizes the chromatin into shorter loops, and allows replication at much shorter intervals along the DNA. Remarkably, these observations also explain an old mystery of why serial nuclear transplantation was so successful for cloning frogs.

Chapter 1. Introduction: conserved steps in eukaryotic DNA replication: Template Origins Origin recognition Pre-initiation Initiation Elongation Termination Chromatid cohesion and segregation. Chapter 2. AAA and ATPases in loading replication factors: ORC and loading of Cdc6 Cdc6 and loading of MCMs RFC and loading of PCNA. Chapter 3. Ring structures and six fold symmetry in replication: Helicases: MCMs WRN, BLM etc Clamps: PCNA Rad9-Rad1-Hus1. Chapter 4: Polymerization mechanisms: Thermodynamics of polymerization Structural insights into polymerization mechanisms fidelity: Replicative polymerase error rates Enhancing polymerase fidelity by protein interactions. Chapter 5. Molecular hand-off mechanisms in replication: Priming polymerization Polymerase switches Okazaki fragment processing: differential affinities for PCNA Methylation and chromatin modification via molecular hand-off mediated through PCNA? RPA in replication/repair. Chapter 6. Co-ordination of nucleases and helicases: Nucleases in replication: conservation of roles and mechanisms Fen1 and Dna2 at Okazaki fragments Stalled forks, helicases and nucleases - BLM, WRN, Exo1 and others. Chapter 7. The switch from replication to repair (and back): Monitoring DNA integrity Stalled replication forks and replisome stability: impact of lesion type Choosing the right polymerase through PCNA modification Polymerase switching over lesions Restarting stalled forks after repair. Chapter 8: Telomeres and the end replication problem: Structure and stability of telomeres Telomerase: structural and drug inhibition studies ALT pathways of telomere maintenance Telomere length and telomere-specific sister chromatid exchange in replicative senescence. Keeping replicated chromatids together until mitosis: Topoisomerases and separating chromatids SMC proteins, claspins and cohesions Attaching chromatids to the mitotic spindle - the role of ORC. Chapter 10. Chromatin modification during replication: Chromatin context and replication patterns Silenced loci and dormant origins DNA methylation during replication Histone modifications (methylation, acetylation, phosphorylation, ubiquitination - CAF1, SIRT1, SNF2/HDACs etc) Nucleosome positioning in relation to origins. Chapter 11. Mittochondrial DNA replication: Template: copy number, structure and origin DNA pol gamma - mode of action Accessory factors Regulation - autonomous or integrated with cell cycle? Co-ordination with damage sensing and repair. Chapter 12. Archaeal paradigms and parallels for eukaryotic DNA replication: Overview: comparison between archaeal and eukaryotic replication proteins and processes Insights into priming from the structure of the primase core Archaeal MCMs as a model for eukaryotic MCMs Structure of the archaeal preRC and stabilization by GINS Protein interactions with PCNA. Chapter 13. Drug targets in DNA replication: Nuclear replication: ORC, MCMs and GINS - markers or targets? Geminin PCNA (p21) and Ciz Polymerases Telomerase Mitochondrial replication targets Malaria and potential novel drug targets.

Retinoic acid (RA) is a powerful differentiation agent. Barrett's oesophagus occurs when duodeno-gastro-oesophageal reflux causes squamous epithelium (SE) tissue to become columnar epithelium tissue by an unknown mechanism. The bile acid lithocholic acid (LCA) competes for the retinoid X receptor retinoid binding site. Hence, RA pathways may be implicated in Barrett's oesophagus.RA activity in tissues and cell lines treated with all-trans retinoic acid (ATRA) with or without LCA was assessed using a reporter. Expression of p21 was determined by real-time PCR in Barrett's oesophagus cell lines with or without LCA. SE and Barrett's oesophagus biopsy specimens were exposed to 100 muM of ATRA or 20 mM of a RA inhibitor, citral, in organ culture for72 h. Characteristics of treated specimens, compared with untreated controls, were analysed by immunohistochemical analysis (cytokeratins (CKs), vimentin) and RT-PCR (CKs). Confocal microscopy assessed temporal changes in co-localisation of CK8/18 and vimentin. Cell proliferation was assessed by bromo-deoxyuridine incorporation and immunohistochemical analysis for Ki67 and p21.RA biosynthesis was increased in Barrett's oesophagus compared with SE (p0.001). LCA and ATRA caused a synergistic increase in RA signalling as shown by increased p21 (p0.01). Morphological and molecular analysis of SE exposed to ATRA showed columnar differentiation independent of proliferation. Metaplasia could be induced from the stromal compartment alone and vimentin expression co-localised with CK8/18 at 24 h, which separated into CK8/18-positive glands and vimentin-positive stroma by 48 h. Citral-treated Barrett's oesophagus led to phenotypic and immunohistochemical characteristics of SE, which was independent of proliferation.RA activity is increased in Barrett's oesophagus and is induced by LCA. Under conditions of altered RA activity and an intact stroma, the oesophageal phenotype can be altered independent of proliferation.

Recent work has clarified how higher eukaryotic cells 'licence' DNA replication precisely once per cycle. An inhibitor, geminin, prevents replication before mitosis by inhibiting the replication factor Cdt1. Degradation of geminin in anaphase allows Cdt1 to promote binding of MCM proteins, and hence DNA replication.